Pacemaking - the generation of spontaneous rhythmic electrical activity in heart and brain cells -- is crucial to life and to normal brain function. "Pacemaker channels," also known as l(f), l(h), or HCN channels, are a specific class of ion channels that underlie this function in many cells. They are members of the voltage- activated ion channel superfamily, but unlike almost all other members of the superfamily, these HCN channels are activated by hyperpolarization. Results from the preceding grant period, and evidence from other labs, support the idea that voltage sensing (movement of a specialized positively-charged protein domain) and gating (opening and closing of the transmembrane pore) in HCN channels are both essentially like that of the other family members. This means that the different behavior of these channels is probably accounted for by a difference in the "coupling" mechanism, whereby movement of the voltage sensor is transduced into opening of the pore. Despite some leads, this is still the most mysterious step in the operation of voltage-gated channels. This proposal will address the coupling question directly, using specific structural constraints -- variable- length bridges between pairs of introduced residues ~ as a tool for learning about the moving parts, their range of motion, and the consequences of constraining this range of motion. In addition to this study of the mechanics of gating and coupling, there will be a quantitative study of coupling energetics in wild-type channels and in a specific set of mutant channels that produce dramatic changes in the gating behavior of the HCN channels. Work from the preceding grant period revealed that some of the "leakage" through wild- type channels that occurs at positive voltages (where the channels should be closed) is due to a voltage- independent mode of channel gating in a subpopulation of HCN channels, which interchanges only slowly with the main voltage-dependent population. Using gating current measurements and tools developed in the last grant period, this project will distinguish this voltage-independent mode from true changes in coupling, which can then be analyzed to learn how natural variants of HCN channels, and mutants, affect the coupling process. The structural constraints and functional studies together will elucidate the physical and energetic mechanism of the key physiological process of coupling between voltage sensors and gates.